Complementary truncations of a hydrogen bond to ribose involved in transition-state stabilization by cytidine deaminase.
The crystal structure of the complex formed between Escherichia coli cytidine deaminase and the transition-state analogue inhibitor 3,4-dihydrouridine [Betts, L., Xiang, S., Short, S. A., Wolfenden, R., & Carter, C. W. (1994) J. Mol. Biol. 235, 635] shows the presence of an H-bond between Glu-91 and the 3'-OH group of substituent ribose, a part of the substrate that is not directly involved in its chemical transformation. To test the contribution of this interaction to transition-state stabilization, Glu-91 was converted to alanine. The mutant enzyme is very much less active than the wild-type enzyme, with a 500-fold increase in Km and a 32-fold reduction in kcat using cytidine as substrate. No change in secondary structure is evident in the circular dichroic spectrum. As measured by kcat/Km, Glu-91 thus appears to stabilize the transition state for cytidine deamination by an overall factor of 1.7 x 10(4), equivalent to -5.8 kcal/mol in free energy. To test the contribution of this interaction in the opposite sense, the 3'-OH group of the substrate was replaced by a hydrogen atom. Comparing 3'-deoxycytidine with cytidine, the native enzyme shows a 17-fold increase in Km and a 400-fold decrease in kcat, indicating that the 3'-hydroxyl group of cytidine stabilizes the transition state for deamination by an overall factor of 6.3 x 10(3), equivalent to -5.2 kcal/mol in free energy, as measured by kcat/Km. After one binding partner has been removed, however, the effect of removing the remaining partner is relatively slight. For the mutant enzyme E91A, removal of the 3'-hydroxyl group from substrate cytidine reduces kcat/Km by a factor of only 3. Complete removal of substituent ribose reduces the wild-type enzyme's kcat/Km by a factor of more than 10(8); thus, substituent ribose, although distant from the site of chemical transformation of the substrate, contributes at least 11 kcal to the free energy of stabilization of the transition state for cytidine deamination, matching the apparent contribution to transition state binding made by the 4-OH group of the pyrimidine ring, which is at the site of substrate transformation [Frick, L., Yang, C., Marquez, V. E., & Wolfenden, R. (1989) Biochemistry 28, 9423].
Carlow
D C
DC
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599-7260, USA.
Short
S A
SA
Wolfenden
R
R
D019943:Amino Acid Substitution / Q000235:genetics; D003564:Cytidine Deaminase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003596:Cytosine / Q000378:metabolism; D003641:Deamination; D003841:Deoxycytidine / Q000378:metabolism; D004795:Enzyme Stability; D018698:Glutamic Acid / Q000235:genetics; D006860:Hydrogen Bonding; D016297:Mutagenesis, Site-Directed; D012266:Ribose / Q000378:metabolism*; D013379:Substrate Specificity
D003841:Deoxycytidine; D018698:Glutamic Acid; D012266:Ribose; D003596:Cytosine; D003564:Cytidine Deaminase